Adjuvant conditioning induces an immunosuppressive milieu that delays alloislet rejection through the expansion of myeloid-derived suppressor cells.

Advances in immunosuppression have been relatively stagnant over the past 2 decades, and transplant recipients continue to experience long-term morbidity associated with immunosuppression regimens. Strategies to reduce or eliminate the dosage of immunosuppression medications are needed. We discovered a novel administration strategy using the classic adjuvant alum to condition murine islet transplant recipients, known as adjuvant conditioning (AC), to expand both polymorphonuclear and monocytic myeloid-derived suppressive cells (MDSCs) in vivo. These AC MDSCs potently suppress T cell proliferation when cultured together in vitro. AC MDSCs also facilitate naïve CD4+ T cells to differentiate into regulatory T cells. In addition, we were able to demonstrate a significant delay in alloislet rejection compared with that by saline-treated control following adjuvant treatment in a MDSC-dependent manner. Furthermore, AC MDSCs produce significantly more interleukin (IL)-10 than saline-treated controls, which we demonstrated to be critical for the increased T cell suppressor function of AC MDSCs as well as the observed protective effect of AC against alloislet rejection. Our data suggest that adjuvant-related therapeutics designed to expand MDSCs could be a useful strategy to prevent transplant rejection and curb the use of toxic immunosuppressive regimens currently used in transplant patients.

Properties of regulatory B cells regulating B cell targets.

Regulatory B cells (Bregs) have shown promise as anti-rejection therapy applied to organ transplantation. However, less is known about their effect on other B cell populations that are involved in chronic graft rejection. We recently uncovered that naïve B cells, stimulated by TLR ligand agonists, converted into B cells with regulatory properties (Bregs-TLR) that prevented allograft rejection. Here, we examine the granular phenotype and regulatory properties of Breg-TLR cells suppressing B cells. Cocultures of Bregs-TLR with LPS-activated B cells showed a dose-dependent suppression of targeted B cell proliferation. Adoptive transfers of Bregs-TLR induced a decline in antibody responses to antigenically disparate skin grafts. The role of Breg BCR specificity in regulation was assessed using B cell-deficient mice replenished with transgenic BCR (OB1) and TCR (OT-II) lymphocytes of matching antigenic specificity. Results indicated that proliferation of OB1 B cells, mediated through help from CD4+ OT-II cells, was suppressed by OB1 Bregs of similar specificity. Transcriptomic analyses indicated that Bregs-TLR suppression is associated with a block in targeted B cell differentiation controlled by PRDM1 (Blimp1). This work uncovered the regulatory properties of a new brand of Breg cells and provided mechanistic insights into potential applications of Breg therapy in transplantation.

Interleukin-27 in liver xenotransplantation: A rational target to mitigate ischemia reperfusion injury and increase xenograft survival.

Transplantation of xenogeneic organs is an attractive solution to the existing organ shortage dilemma, thus, securing a clinically acceptable prolongation of xenograft survival is an important goal. In preclinical transplantation models, recipients of liver, kidney, heart, or lung xenotransplants demonstrate significant graft damages through the release of pro-inflammatory molecules, including the C-reactive protein, cytokines, and histone-DNA complexes that all foster graft rejection. Recent studies have demonstrated that mitigation of ischemia reperfusion injury (IRI) greatly improves xenograft survival. Organ IRI develops primarily on a complex network of cytokines and chemokines responding to molecular cues from the graft milieu. Among these, interleukin 27 (IL-27) plays an immunomodulatory role in IRI onset due to graft environment-dependent pro- and anti- inflammatory activities. This review focuses on the impact of IL-27 on IRI of liver xenotransplants and provides insights on the function of IL-27 that could potentially guide genetic engineering strategies of donor pigs and/or conditioning of organs prior to transplantation.

Detection of alloreactive T cells from cryopreserved human peripheral blood mononuclear cells.

Precise analyses of alloreactive T cell phenotype and function can inform both the nature and intensity of adaptive responses to transplant antigens. However, alloreactive T cells are sparse and difficult to detect, particularly in cryopreserved peripheral blood mononuclear cells (PBMCs) and from hypo-responsive individuals. An assay to identify and phenotype alloreactive cells would be particularly valuable, especially for multi-center clinical trials that often store frozen samples for batch analysis. Herein we demonstrate consistent and reproducible alloreactive T cell detection in cryopreserved PBMC following a short-term mixed lymphocyte reaction (MLR). The inherent background expression levels of activation markers on responder T cells were minimized by including a resting period prior to the assay. Stimulator cells were activated before inclusion in the MLR by addition of CD40L and IL-4. The time frame and markers to identify and phenotype alloreactive T cells following stimulation were optimized using short term co-cultures. We defined subsets of CD4+ and CD8+ T cells co-expressing CD69 and either CD154 or CD137 following allostimulation as alloreactive, and further phenotyped these cells with a variety of surface markers such as PD-1, LAG-3, and TIM-3. This assay may allow for the monitoring of donor-specific T cells in transplant recipients with longitudinally collected and cryopreserved PBMCs and provide a useful tool to identify biomarkers associated with tolerance. These biomarkers may add to mechanistic insights in immune recognition of transplanted tissues and/or cells.

Rap GTPase Interactor: A Potential Marker for Cancer Prognosis Following Kidney Transplantation.

Post-transplant (post-Tx) kidney cancer has become the second-highest cause of death in kidney recipients. Late diagnosis and treatment are the main reasons for high mortality. Further research into early diagnosis and potential treatment is therefore required. In this current study, through genome-wide RNA-Seq profile analysis of post-Tx malignant blood samples and post-Tx non-malignant control blood samples (CTRL-Tx), we found Rap GTPase Interactor (RADIL) and Aprataxin (APTX) to be the most meaningful markers for cancer diagnosis. Receiver operating characteristic (ROC) curve analysis showed that the area under the curve (AUC) of the RADIL-APTX signature model was 0.92 (P < 0.0001). Similarly, the AUC of RADIL alone was 0.91 (P < 0.0001) and that of APTX was 0.81 (P = 0.001). Additionally, using a semi-supervised method, we found that RADIL alone could better predict malignancies in kidney transplantation recipients than APTX alone. Kaplan-Meier analysis indicated that RADIL was expressed significantly higher in the early stages (I and II) of kidney, liver, stomach, and pancreatic cancer, suggesting the potential use of RADIL in early diagnosis. Multivariable Cox regression analysis found that RADIL together with other factors (including age, stage III, stage IV and CD8+ T cells) play a key role in kidney cancer development. Among those factors, RADIL could promote kidney cancer development (HR > 1, P < 0.05) while CD8+ T cells could inhibit kidney cancer development (HR < 1, P < 0.05). RADIL may be a new immunotherapy target for kidney cancer post kidney transplantation.